Systematic analysis and expression profiles of TCP gene family in Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn.) revealed the potential function of FtTCP15 and FtTCP18 in response to abiotic stress

Background As transcription factors, the TCP genes are considered to be promising targets for crop enhancement for their responses to abiotic stresses. However, information on the systematic characterization and functional expression profiles under abiotic stress of TCPs in Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn.) is limited. Results In this study, we identified 26 FtTCPs and named them according to their position on the chromosomes. Phylogenetic tree, gene structure, duplication events, and cis-acting elements were further studied and syntenic analysis was conducted to explore the bioinformatic traits of the FtTCP gene family. Subsequently, 12 FtTCP genes were selected for expression analysis under cold, dark, heat, salt, UV, and waterlogging (WL) treatments by qRT-PCR. The spatio-temporal specificity, correlation analysis of gene expression levels and interaction network prediction revealed the potential function of FtTCP15 and FtTCP18 in response to abiotic stresses. Moreover, subcellular localization confirmed that FtTCP15 and FtTCP18 localized in the nucleus function as transcription factors. Conclusions In this research, 26 TCP genes were identified in Tartary buckwheat, and their structures and functions have been systematically explored. Our results reveal that the FtTCP15 and FtTCP18 have special cis-elements in response to abiotic stress and conserved nature in evolution, indicating they could be promising candidates for further functional verification under multiple abiotic stresses. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-022-08618-1.


Background
Transcription factors usually contain DNA-binding domains, transcription regulation domains, oligomerization sites and nuclear localization signals. Some domains are used to bind DNA, and some are used to activate transcription [1]. A total of 58 transcription factor families have been identified from 156 plant genome sequences [2]. Many studies have shown that identifying key transcription factors provides new insights into how plants respond to abiotic stresses at the cellular and molecular levels [3][4][5][6][7][8]. Abiotic stress factors or their different combinations can affect the yield and quality of crops on cultivated land worldwide by influencing physiological and biochemical processes within the plant [9][10][11]. In consideration of global climate change, transcription factors are promising targets for crop enhancement because they are key regulatory switches that control gene expression in various organic and metabolic processes [12].
The transcription factor TCP gene family was originally identified and named based on the characteristics of the first three family members of Zea mays (L.) Sp. (TB1), Antirrhinum majus L. (CYC), and Oryza sativa L. (PCF) [13]. The TCP gene family contains highly conserved and non-canonical TCP domains with a basic helix-loophelix structure. These domains play a role in proteinprotein dimerization and DNA binding [14,15]. The TCP family is plant-specific and can regulate plant growth, including seed germination and seedling establishment [6], spikelet meristem [16], and branching [17]. Online interaction network prediction shows that the TCP gene family also participates in regulating and synthesizing numerous protein complexes involved in various biological processes such as plant hormone pathways, cell life cycles, and environmental stress responses [18]. Many studies have confirmed that the TCP family is involved in plant responses to abiotic stresses. When the OsTCP19 gene is over-expressed in Arabidopsis, it can enhance plant tolerance to drought and heat during seedling and maturity stages [19]. Additionally, down-regulation of AsTCP14 in the Creeping bentgrass (Agrostis stolonifera L.) can improve plant resistance to drought and salt stresses [20]. TCP transcription factors inhibit the formation of trichomes on the side of Arabidopsis (Arabidopsis thaliana (L.) Heynh.) cotyledon, thus affecting the barrier function of these trichomes against biotic and abiotic stresses on the plant [21].
Buckwheat is a pseudo-cereal crop with a long cultivation history and incomparable nutritional characteristics and is one of the potential research objects to realize the diversification of food resources [22][23][24]. Buckwheat is cultivated and consumed worldwide and has received widespread attention for its synthesis by many substances with health functions and active medicinal ingredients [24][25][26]. The buckwheat is usually planted in habitats with low rainfall, low temperatures, and high altitudes [27,28]. In addition, sufficient light and ultraviolet radiation, for example, high UV-B doses, can affectthe physiological and biochemical processes of plants [29][30][31], contributing to unusual energy conversion efficiency and plant tissue function [32].To sum up, the growth, yield, and quality of buckwheat are severely threatened by abiotic stress factors or their combinations. To date, many transcription factors in Tartary buckwheat have been well-studied. Some of these transcription factors have been identified to be associated with Al stress [33] and salt stress [34]. However, information on the identification, expression analysis, and potential functions under abiotic stresses of TCP transcription factors in Tartary buckwheat is limited.
In this study, the genome-wide identification of 26 TCPs in Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn.) was carried out. Subsequently, FtTCP15 and FtTCP18 were compared with other members of the FtTCP gene family in terms of evolutionary relationship, gene structure, conservative motifs, and cis-acting elements. Next, expression profiles of selected 12 FtTCP genes under multiple abiotic stresses, including cold, dark, heat, salt, UV, and waterlogging (WL), were analyzed by qRT-PCR, FtTCP15 and FtTCP18 were found to be highly expressed under multiple treatments. Correlation coefficient analysis of gene expression levels, tissuespecific expression, and interaction network prediction was conducted. Then, subcellular localization through laboratory methods demonstrated that both FtTCP15 and FtTCP18 were localized in the nucleus. Our work provides a comprehensive framework for how FtTCPs respond to abiotic stresses and affords valuable clues for further functional verification of FtTCP15 and FtTCP18 as transcription factors in Tartary buckwheat to improve the adaptability under multiple abiotic stresses.

The 26 FtTCP genes identified in the Tartary buckwheat genome are unevenly distributed on 8 chromosomes
In this paper, 26 FtTCPs from the Tartary buckwheat genome were identified and named from FtTCP1 to FtTCP26 according to their position on the chromosome (Fig. 1). The distribution and arrangement information of genes on chromosomes in the latest Tartary buckwheat gene bank were used for chromosome distribution maps. Figure 1 shows that the 26 FtTCP genes of Tartary buckwheat are unevenly distributed on 8 chromosomes, with 2 to 6 genes on each chromosome. The Ft8 has the largest number of FtTCP genes (6 genes), while Ft1, Ft5, and Ft7 have the least number of FtTCP genes (2 genes). There are 3 genes on Ft2 and Ft6, and 4 genes on chromosomes Ft3 and Ft4. Additionally, the two genes on Ft1 are on the reverse chain, and both the two genes on Ft7 are on the forward chain. The genes on the remaining chromosomes are also in forward or reverse chains. Table S1 shows detailed information about these FtTCPs, including the gene name, sequence ID, group, length of protein (PL), molecular weight (MW), the isoelectric point (pI), genome location, and predicted subcellular location (W). The length of the identified FtTCP gene-encoded proteins ranges from 176 aa (FtTCP2) to 475 aa (FtTCP3), with an average of 285.8 aa. The range of MW is from 19.17 kDa (FtTCP2) to 51.04 kDa (FtTCP3), with an average of 31.09 kDa. The theoretical pI also differs greatly from 4.93 (FtTCP20) to 9.71 (FtTCP19) with an average of 7.05. According to the subcellular localization prediction, 21 FtTCP genes have the highest probability of being in the nucleus, and 5 FtTCP members are only located in the nucleus. Additionally, the group and chromosome location of the identified FtTCP genes were provided.

The phylogenic relationship and conserved features predict the potential functions of FtTCP15 and FtTCP18
In order to understand the phylogenetic relationship among TCP genes of Tartary buckwheat, Arabidopsis, and rice, the full-length sequence of TCP proteins with known functions was selected to draw a rootless phylogenetic tree (Fig. 2). The numbers of proteins from Tartary buckwheat (FtTCP), Arabidopsis (Arabidopsis thaliana (L.) Heynh.) (AtTCP), and rice (Oryza sativa L.) (OsTCP) were 26, 24, and 22, respectively. The names used for the AtTCP in Arabidopsis and OsTCP in rice were based on the previous study [35]. The result shows that all 72 TCP members are classified into Class I and Class II subfamilies. Class I contains the PCF group, and Class II contains two subclades (CYC/TB1 and CIN). The phylogenetic tree shows that 36 TCPs belong to the PCF clade, 26 to CIN, and 10 to CYC/TB1. The results show that AtTCPs and OsTCPs in the phylogenetic tree fall into the same Class or subclade consistent with the finding in the previous study [35]. All the 26 FtTCPs are classified into three clades, with 12, 6, and 8 in PCF, CYC/TB1 and CIN, respectively. The distribution of the TCP genes in each subfamily is random and uniform. For example, there is only 1 from rice, 6 from Tartary buckwheat, and 3 from Arabidopsis among the 10 CYC/TB1 TCP genes.
As shown in Fig. 2 [36] and tomatoes [37]. In particular, FtTCP15 and FtTCP23 are isolated from each other, and their evolutionary relationships are closely related to FtTCP6 and AtTCP10. In contrast, FtTCP18 is closely related to AtTCP8, OsTCP19, and OsTCP21, indicating that these genes in the same cluster may have similar functions in response to abiotic stresses. Therefore, FtTCP15 and FtTCP18 may have the same function as AtTCP10 and OsTCP19.
As shown in Fig. 3, the structural features and conserved motifs have been performed to further study TCP genes. The analysis provides a reference to analyze the evolutionary relationship and the structural diversity of members in each subfamily and to fully understand conserved motifs and gene structure of the TCP family. As shown in Fig. 3b, there are 15 conserved motifs (motif 1-motif 15) of the FtTCP protein predicted by the online MEME analysis tool. The different colored boxes in the legend correspond to distinct motifs. The logo and name of the query motifs are displayed in Table S2. Motif 1 and Motif 2 are present in nearly all FtTCP proteins and Motif 1 is included in every protein. However, other motifs exist in less than half of the proteins. Many motifs, such as motifs 6, 9, 14, and 15, shared by the class II (CYC/TB1 and CIN) TCP family, are restricted to specific groups. The CIN group genes only share the last three motifs. Additionally, Motif 13 only exist in FtTCP3 and FtTCP16 (belonging to PCF Group), and Motif 6 is only found in FtTCP7 and FtTCP8 (belong to CYC/TB1). FtTCP24 only has Motif 1.
The structural features of the FtTCP gene family, including the arrangement and quantity characteristics of exons and introns, are shown in Fig. 3c. Gene family Among the remaining 10 genes with introns, 8 belong to Class II (6 belong to CIN Group, 2 belong to CYC/ TB1 Group). The CIN group contains the most members of introns compared with the other two groups. The FtTCP5 belonging to CIN has the longest exon. Moreover, the number of introns contained in the remaining 10 genes ranges from 1 to 3, indicating little difference among these members. A total of 22 FtTCP members contain exons, accounting for 84%. The genes with exons in the Arabidopsis TCP family account for about 82% [35]. The results indicate that buckwheat and Arabidopsis have similar gene conservatism. Both FtTCP15 and FtTCP18 have no introns, indicating that they are highly conservative in response to abiotic stress. FtTCP15 contains Motifs 1,2,8,9,14,15; FtTCP15 contains 2 Motif 5, and Motifs 1, 2, 7. Their specific conserved motifs can be used as the targets for further studies on the molecular mechanisms related to the stress response.

Segmental duplications are the main force in the molecular evolution of FtTCP gene amplification
Gene duplication is the main force that promotes the amplification of gene families and generates new functions of genes. The analysis of replication events (Fig. 4) shows that only segmental duplication events happen in the FtTCP gene family of the Tartary buckwheat genome, and there are no tandem duplication events. As shown in Fig. 4, there are 10 pairs of segment duplication events in the FtTCP gene family. Ft8 and Ft3 have 4 segmental duplication gene pairs. There are 3 segmental duplication gene pairs that occur on Ft1, Ft2, and Ft4, simultaneously. The remaining Ft5 and Ft6 have 2 and 1 segmental duplication gene pairs, respectively. However, no segmental duplication gene pairs occur on the Ft7. As for the subfamily, there are 6 repeated events in Class I (PCF) subfamily. A total of 10 segmental duplication events occurred in CIN subfamily with the highest number, and 4 segmental duplication events occurred in the CYC/TB1 subfamily. There are 14 segmental duplications events in Class II (CIN and CYC/TB1) in all. Segment duplication events can promote the evolution of the FtTCP gene family. Intraspecific collinearity was found between FtTCP15 and FtTCP6, but no intraspecific collinearity was found for FtTCP18.   CIN group, 7 are classified into the PCF group, and only 1 belongs to CYC/TB1. Additionally, the FtTCP15 located on Ft5 has only one interspecific collinearity gene pair with soybean, and FtTCP18 located on Ft6 has one pair with tomato, soybean, and apple, respectively, suggesting that they are relatively conservative during evolution.

Analysis of cis-acting elements of FtTCPs reflects their potential function in stress response
Cis-acting elements refer to specific DNA sequences in series with structural genes and bind sites for transcription factors. Cis-acting elements regulate the precise initiation and transcription efficiency of gene transcription by binding to transcription factors. In this work, an upstream 2000 bp region was selected to analyze the ciselements of the FtTCP genome sequence ( Fig. 6; Table  S3; Table S4).

The response of the FtTCP family to abiotic stress is spatio-temporal, and the interaction network is predicted
A total of 12 FtTCPs were selected randomly for expression analysis under multiple abiotic stresses, including cold, dark, heat, salt, UV, and WL. A histogram of the expression of these genes in different tissues after stress at different time points was drawn, and correlation analysis was performed. As shown in Fig. 7 and Figure S1, the expression patterns of FtTCPs under different abiotic stress conditions have time and space specificity, and many genes have obvious up-regulation or down-regulation trends.
FtTCP11 ( (FtTCP2, FtTCP18, and FtTCP19) were restricted by WL, UV, salt, dark, cold, and heat stresses, respectively. Among them, 5 FtTCPs (FtTCP8, FtTCP12, FtTCP24, FtTCP25, and FtTCP26) were activated by the specific stress. Several genes were significantly up-regulated in different stress environments at two time points of stress and in three different tissues. The expression profiles of the selected genes under abiotic stress revealed that FtTCP18 was generally responsive to different abiotic stresses, and FtTCP15 was responsive to WL, UV, and salt stress. We are interested in exploring the molecular mechanisms of FtTCP15 and FtTCP18 in response to abiotic stresses. Therefore, we conducted a comprehensive study on the evolution of the FtTCP gene family to explore the particularity of FtTCP15 and FtTCP18.
The correlation analysis of gene expression under different abiotic stress conditions ( Figure S1) demonstrates that the maximum correlation coefficient is 0.81 (between FtTCP2 and FtTCP8), and the minimum is -0.09 (between FtTCP8 and FtTCP9). These correlation coefficients prove that these genes have a certain connection when performing their functions. FtTCP15 and FtTCP18 are representatives in expression profile analysis under several stresses. A basic overview of multiple potential regulatory mechanisms of FtTCPs can be obtained through various expression patterns in different organizations. In addition, tissue-specific expression analysis of the selected genes in Tartary buckwheat was conducted in the seedling stage (six-week-old) under natural conditions. As shown in Fig. 8a, TCP genes of Class I are highly expressed in the leaf and root. For example, FtTCP18 and FtTCP24 are highly expressed in the leaf, while FtTCP11 and FtTCP19 are highly expressed in the root. The genes from Class II are expressed in all tissues. A particular concern is that FtTCP15 is highly expressed in the root, while the FtTCP18 is highly expressed in the leaf, indicating their different functions in responding to stresses in different tissues. Interaction network prediction is significant for studying the function of genes. Interaction prediction data were obtained from STRING (https:// string-db. org/) and visualized using Cytoscape software (Table S5; Table S6). As shown in Fig. 8b, the size of the red circle can intuitively represent interaction times between proteins. The FtTCP24 alone interacts with the TBD4 and AT3G48420, indicating that FtTCP24 may have a different function. FtTCP15 and FtTCP18 interact with multiple proteins in the interaction network, and the number of FtTCP15 and FtTCP18 interacting with other proteins is 41 and 44, respectively (Table  S6).

Subcellular localization showed that FtTCP15 and FtTCP18 localized in the nucleus
Transcription factors can regulate the transcription process of genes in the nucleus. To a certain extent, transcription factors can be determined by their subcellular localization. Figure 9 shows the subcellular localization analysis of the FtTCP15 and FtTCP18. It is found that both FtTCP15 and FtTCP18 can be localized in the nucleus, which is consistent with the general understanding of the properties of transcription factors [40]. FtTCP15 also shows weak fluorescence signals in the cytoplasm, suggesting many exceptional cases for subcellular localization of transcription factors.

TCP gene family in Tartary buckwheat is highly conserved
The phylogenetic analysis and sequence alignment of Tartary buckwheat, Arabidopsis, and rice indicate that all 26 FtTCPs can be classified into Class I (PCF) and Class II (CIN and CYC/TB1) (Fig. 2). Genes with close positions in the rootless phylogenetic tree have similar domains, which indicates that their functions are similar. For instance, the FtTCP15 is closed to the AtTCP10, which is one of the members of miR319-regulated class II TCPs and regulates multiple physiological processes, including jasmonic acid (JA) biosynthesis [41]. The OsTCP19 gene belonging to PCF (Class I) has been reported to play a role in modulating abscisic acid pathways to enhance the tolerance to drought and heat during the seedling and maturity stages [19]. The FtTCP18 may have the same function.
Among the two subfamilies, the Class I subfamily has more genes without introns in structure. Moreover, the same group of FtTCP members displays relatively conserved motif compositions and a similar exon-intron organization (Fig. 3), indicating that the FtTCPs have close evolutionary relationships and similar functions to the TCP genes in plants. The similarity of gene structure provides further evidence for the credibility of subfamily classification. Introns can increase the length of genes and the frequency of gene recombination. In this study, 16 FtTCPs, including the FtTCP15 and FtTCP18, have no introns, accounting for 62%. Genes with few introns tend to respond to stress more rapidly [42], and the introns-less genes of FtTCPs may facilitate the response to environmental changes. Additionally, specific conserved motifs are also one of the factors that contribute to the potential function. For example, Motif 5 appears twice in FtTCP15 and may have a special significance for the function of FtTCP15.
Moreover, the expansion in the evolution of the TCP family in Tartary buckwheat is due to segmental duplications rather than tandem duplications (Fig. 4). This fact is consistent with previous research results in Arabidopsis, cotton [43], and grape [44]. The results show that segment duplication is the main driving force for the evolution and expansion of the TCP gene family, coinciding with the relatively high conservation of the TCP gene family. In addition, more orthologous gene pairs from the CIN group were identified among Tartary buckwheat, rice and Arabidopsis (Fig. 5), showing that TCP genes in these plant species originated from a common ancestor during evolution and the genes in the CIN group have the same function.
In conclusion, the lack of introns in gene structure and the conserved nature in evolution endow the functional similarity and stability of TCPs across species, which provides an important genetic basis for further studying the function of TCPs in the response of Tartary buckwheat to abiotic stresses.

Cis-elements indicate the FtTCP15 and FtTCP18 play a unique role in the response of Tartary buckwheat to abiotic stresses
TCP transcription factors usually mediate the transmission of hormone signals, regulate the activity of phytohormone [45], and inhibit the promoting effect of gibberellin [46] and the biosynthesis of auxin [47] in the plant. Recent studies in Arabidopsis have suggested that members of the TCP family can form response modules with other genes to jointly regulate methyl jasmonate homeostasis [41], and thus may affect plant responses to abiotic stresses. Therefore, the FtTCPs can directly or indirectly affect cellular functions of plants, thereby affecting the plants' growth, development, and response to abiotic stresses.
In this paper, the proportion of cis-elements responding to stress is 17% in Tartary buckwheat (Table S3, S4). The relatively high number of two cis-element promoters (anaerobic induction and anoxic specific inducibility) would result in the high expression of TCP family members in the anaerobic environment, such as WL. Typically, WL is probably caused by artificial irrigation, drainage, and excessive non-human rainfall. An oxygendeficient state will occur when plants are in a flooded state for a long time [48]. These results provide insights into the molecular mechanism of the response of FtTCP genes, especially FtTCP15 and FtTCP18, to WL (Fig. 7). Particularly, both FtTCP15 and FtTCP18 were significantly activated by WL and UV treatments (Fig. 2). Moreover, all light-responsive elements were found to be abundant in all FtTCPs. This result may be related to the biological clock regulation function of the TCP gene family previously reported [49]. In addition, we speculated that the light response elements are also related to the UV stress response. Ultraviolet radiation can affect plant growth and morphogenesis, especially in places with sufficient light, and high doses of UV-B, which can affect physiological and biochemical processes [31].
These results demonstrate that the TCP gene family may be involved in plant responses to abiotic stresses by regulating different biological processes such as direct response, hormonal regulation, and indirect response to abiotic stresses through morphogenesis. For example, the COM1 protein, which is homologous to the BDI1 TCP regulator in sorghum and Brachy podium, has been reported to adjust the shape of the spike inflorescence [50].

The FtTCPs act as important nodes in the interaction network in response to abiotic stresses
In recent years, several works have demonstrated that transcription factors could be promising targets for improving adaptability under environmental stress. In this work, 26 FtTCP genes were identified from the Tartary buckwheat genome. Under six abiotic stresses, the selected 12 genes in the root, stem, and leaf responded to different stress environments and had different expression patterns in different tissues and at different times. Among the 12 FtTCPs, five genes were activated by the specific stress, and the correlation coefficients of gene expression levels under abiotic stress were calculated ( Fig. 7; Figure S1). The TCP family in soybean exhibited similar response trends under different abiotic stresses [51]. In this work, FtTCP2 and FtTCP8 showed the highest correlation of expression levels under abiotic stress and both of them were highly expressed in the stem (Fig. 8a). The FtTCP18 participated in multiple stress responses. Additionally, FtTCP15 was highly up-regulated under WL and UV environments, with the root, stem, and leaf expressed for 24 h.
Transcription factors, such as homo-dimers and hetero-dimers, often build combinations or interact with other proteins rather than acting alone to perform their complex biological functions [8]. Moreover, studies have shown that TCPs belonging to the CIN group can inhibit MYB gene-related transcripts and protein complexes at the transcription and protein levels [21], indicating that TCP members can form a regulatory network through interactions with other genes. The TCPs module, consisting of Class I and Class II TCP proteins, has been reported to manipulate important development processes, including hormone homeostasis in Arabidopsis [41]. In this study, FtTCPs have a high interaction frequency in the interaction network. For example, the nodes of FtTCP15 and FtTCP18 are 41 and 44, respectively. These results indicated that FtTCPs have an

The regulation role of FtTCPs as transcription factors
Subcellular localization shows that FtTCP18 is located in the nucleus (Fig. 9). Additionally, the determination of the subcellular localization of FtTCP15 was in the nucleus and cytoplasm. Some studies also indicated that the subcellular localization of transcription factors is not in the nucleus or incompletely in the nucleus [52,53]. There are many possible reasons for this phenomenon. Firstly, the results of subcellular localization analysis with different receptor materials are probably inconsistent. For example, bHLH039 is mainly localized in the cytoplasm of tobacco leaf cells. In contrast, the nucleus and cytoplasm of Arabidopsis protoplasts have obvious signals [53]. Secondly, some treatments can change the location of transcription factors. As a transcription factor, BZR1 is located in the cytoplasm without any treatment. After Brassinosteroids (BRs) treatment, BZR1 is recruited into the nucleus because the original components in the cytoplasm have been transferred [52].
In conclusion, we proved that the FtTCP family plays a biological function as transcription factors in buckwheat by laboratory methods. For further study, various methods can be used to verify the transcriptional activity of FtTCPs. For example, a yeast experimental system can be used to analyze the transcriptional activation activity of NAC family members [54].

Further studies on the function and molecular mechanism of FtTCP15 and FtTCP18 response to abiotic stresses in Tartary buckwheat are imperative
As an important pseudo-cereal crop, Tartary buckwheat will also face severe challenges to growth in the natural environment. As the global climate worsens, abiotic stress factors such as WL, UV, salt, dark, heat, cold and/ or their combinations pose a serious threat to the growth, yield, and quality of crops. Additionally, TCP transcription factors can transform various endogenous and environmental signals and provide the most comfortable conditions for plant growth [55]. It is credible to hypothesize the manipulation of candidate FtTCP members in Tartary buckwheat with multiple stress adaptability.
In this study, FtTCP15 and FtTCP18 can be regarded as promising candidates for improving the adaptability of abiotic stress in Tartary buckwheat for their molecular characteristics, response to multiple abiotic stresses, and high correlation with other proteins. It is imperative to further study their function by molecular biology techniques. For example, the role of transcription factors in response to abiotic stresses can be determined by studying the binding of transcription factors and promoters using the dual-luciferase gene detection test and by studying the interaction of transcription factors with other proteins through subcellular colocalization tests. Conclusions about the analysis of the clear transcription network, identification of target genes, clarification of the connection with hormones and/or metabolic synthesis, and transcription modification [56] can elucidate the systemic functions of FtTCPs transcription factors in response to stress in Tartary buckwheat.

Conclusions
Many studies have reported that the TCP gene family is vital to abiotic stress response in plants. In this work, 26 FtTCPs were identified in Tartary buckwheat and unevenly distributed on eight chromosomes. Further studies on evolutionary relationships, conservative motifs, gene structure, and cis-elements of the FtTCP gene family have been conducted. Under multiple abiotic stress treatments, the expression levels of the 12 selected FtTCPs were correlated, and the expression patterns of these genes tended to be spatio-temporal specific. The interaction network prediction of FtTCPs with many proteins indicated that FtTCPs are essential in the genetic manipulation response to abiotic stress. In addition, FtTCP15 and FtTCP18 are responsive to multiple abiotic stresses. The subcellular localization showed that both FtTCP15 and FtTCP18 localized in the nucleus, and the properties of FtTCPs as transcription factors are confirmed by laboratory methods. The results demonstrated the conserved nature in the evolution of the TCP gene family in Tartary buckwheat and suggested that the TCP gene family is functionally conserved in different species. The results also showed that the TCP gene family may be involved in plant responses to abiotic stresses by regulating different biological processes. Therefore, further studies on the functions of FtTCP15 and FtTCP18 by molecular biology techniques are feasible and imperative.

Bio-informatics data of TCP genes in Tartary buckwheat
In this study, two approaches were used to identify the TCP family in the Tartary buckwheat genome. The Hidden Markov Model (HMM) profile and the whole genome sequence information of Tartary Buckwheat were obtained from TBGP (Tartary Buckwheat Genome Project; http:// www. mbkba se. org/ Pinku1/). These sequences were then combined to compare the model with the TCP domain (PF03634) from the Pfam protein family database (http:// pfam. xfam. org/) to ensure consistency. Two BLASTp methods were used to identify FtTCP members. First, all possible FtTCP proteins referring to TCP protein sequences were identified with BLASTp. Then, candidate FtTCP proteins containing the TCP domain were obtained by removing the redundant sequences using PFAM and SMART programs. Second, BLASTp in NCBI was used to verify whether all candidate FtTCPs were members of the TCP family. Finally, 26 FtTCPs were identified. As shown in Table S1, the coding sequence (CDS) length, isoelectric point (pI), and amino acid numbers were analyzed. Conserved domains for protein were analyzed using SMART (http:// smart. embl-heide lberg. de/). The subcellular localization was predicted using the ExPasy website (http:// web. expasy. org/ protp aram/).

Genome-wide characteristic analysis of the FtTCP gene family
The distribution of FtTCPs on eight Tartary buckwheat chromosomes was obtained using CIRCOS software (Version 0.69).
Then, the TCP protein sequence was obtained from the UniProt database (https:// www. unipr ot. org). The full-length amino acid sequences and TCP domain sequences, 26 from Tartary buckwheat (FtTCP), 24 from Arabidopsis (Arabidopsis thaliana (L.) Heynh.) (AtTCP), and 22 from rice (Oryza sativa L.) (OsTCP), were used to generate an unrooted phylogenetic tree. The Arabidopsis TCP protein sequence was downloaded from The Arabidopsis Information Resources (TAIR) database (http:// www. arabi dopsis. org). The TCP protein sequences for multiple amino acid sequence alignments in three plants were analyzed, and the phylogenetic tree was built using the neighbor-joining (NJ) method of MEGA 7.0 Software, Geneious R11 with a BLOSUM62 matrix, and the Jukes Cantor substitution model. We set the global alignment with free end gaps and a bootstrap value of 1000.
The exon/intron structures of FtTCPs were generated with TBtools software [57]. The differences in the conserved protein motifs in Tartary buckwheat TCP proteins were compared using the protein conserved motif online search program MEME (http:/ meme. nbcr. net/ meme/ intro. html). The motif breadth was set as 6 to 200 amino acids (aa). A total of 15 motifs (named Motif 1 to Motif 15) were selected for analysis (Table S2).
Multiple collinear scanning toolkits (MCScanX) with an E-value of 1e-10 were used to study the gene duplication events in The sequence regions 2000 bp upstream of FtTCP family members were extracted from the Tartary buckwheat genome as putative promoters. Then, the cis-acting elements on TBtools software (Version 1.0 +) [57] were predicted according to these putative promoters (Table S3;  Table S4).

Plant growth and treatments
The seeds of Tartary buckwheat were cultured in plastic pots in Guiyang, Guizhou Province, China. The cultivation environment was a 14/10 h photoperiod (daytime, 06:00-20:00) cycle with about 80% humidity at 22 °C. In order to analyze the expression profiles of FtTCP genes in roots, stems, and leaves in response to stress, 6-weekold seedlings were subjected to multiple abiotic stresses, including cold (low temperature, 4 °C), dark, heat (40 °C), salt (salinity, 200 mM NaCl) [34], UV radiation (70 µW/ cm 2 , 220 V, 30 W), and WL (water inundating the surface of the earth with ddH 2 O). Under all treatments, samples were harvested with three biological replicates at 0, 2, and 24 h, then immediately frozen in liquid nitrogen, and stored at -80 °C for RNA extraction. Seedlings without stress treatment were the control. Moreover, seedlings grown in natural conditions were collected for tissuespecific analysis of genes with three biological replicates.

RNA extraction, expression analysis by qRT-PCR, and interaction network prediction
In this work, to ensure that the response of genes contained in each subfamily to stress could be observed, 2 to 5 genes were randomly selected from each group (12 genes in total) for expression analysis. Total RNA was isolated from the sample using the RNAiso reagent Trizol (Invitrogen) according to the manufacturer's instructions. Then the potential genomic DNA contamination was removed from RNA using RNase-free DNase I (TaKaRa). The first-strand cDNA was synthesized using the Prime Script RT enzyme mix (Takara). Moreover, the QTower3G instrument (Analytik Jena AG) was used for the qPCR experiment, with each 10 µL amplification reaction containing 5 µL of 2 × PCR MIX (Roche), 0.1 µL of each primer (up 50 pM/µL, down 50 pM/µL), 3.8 µL of H 2 O, and 1 µL of cDNA template. The PCR cycle program included initial denaturation (95 °C/10 min) and 40 cycles of 94 °C for 10 s, 60 °C for 20 s, and 60 °C for 20 s. The gene-specific primers (Table S7) were designed based on the nucleotide sequence of the TCP gene in Tartary buckwheat using Primer 5.0 software (http:// frodo. wi. mit. edu/). The Tartary buckwheat FtH3 gene was employed as an internal reference sequence with the same program [58]. Three technical repeats were performed for each biological replicate, and the relative expression levels of each gene under abiotic stress were calculated by the 2 -∆∆Ct method [59] compared with the control gene. The relative expression levels for tissuespecific analysis of each gene were calculated by the 2 -∆Ct method. Finally, variance analysis and histogram were conducted on all data, and Duncan's multiple range test (P < 0.05; n = 3) was performed using SPSS (Version 25.0) and the Origin software (Origin Lab Corporation, Northampton, Massachusetts, USA) (Version 8.0) statistics program. Data correlation was analyzed using the RStudio (https:// www. rstud io. com/) "corrplot" package. The analysis of heatmaps visualizing expression was performed using TBtools software (Version 1.0 +) [57].
The interaction network between the TCP family with known proteins and predicted proteins was predicted using the String (https:// string-db. org/) for online protein-protein interaction prediction. The prediction included direct physical interactions between proteins and indirect protein functional relevance interactions. After downloading, relevant data of the prediction (Table  S3; Table S4) were visualized using Cytoscape (Version 3.8.2) (https:// cytos cape. org/ downl oad. html).

Subcellular localization
According to the expression levels of genes under abiotic stress, FtTCP15 and FtTCP18 were found to be generally responsive to multiple abiotic stresses. Moreover, we are interested in further exploring the molecular mechanisms of FtTCP15 and FtTCP18.
First, wild-type Colombian Arabidopsis seedlings were cultured in greenhouses at 25 °C for protoplast preparation. The first step: After 25 days of incubation (no bolting), some leaves were soaked in the enzymolysis solution (Cellulase R10, 1.5%; Macerozyme R10, 0.75%; Mannitol, 0.6 M; MES (pH 5.7), 10 mM; H 2 O, constant volume of 10 mL), and then left to stand at 24 °C for 4 h. The second step: The treated samples were filtered through a 40 μm strainer and centrifuged at 300 rpm for 3 min to remove the supernatant. Then samples were washed twice with 10 mL of pre-cooled W5 solution (NaCl (58. Second, the coding regions of FtTCP15 and FtTCP18 were amplified by PCR and fused to the pBWA(V)HSccdb-GLosgfp (Wuhan RiORUN Co., Ltd, China), which was modified by the pCAMBIA1302 expression vector. Using pBWA(V)HS-ccdb-GLosgfp as control, pBWA(V) HS-FtTCP15-GLosgfp and pBWA(V)HS-FtTCP18-GLosgfp were transferred into the Arabidopsis protoplasts prepared from leaves [60]. The 100 μL of protoplast suspension was mixed with a 20 μL plasmid of the target DNA plasmid. Simultaneously, PEG4000 solution (120 μL) equal to the total volume of DNA and protoplast was added, gently mixed, and left to stand at room temperature for 30 min. Finally, the reaction was terminated with 1 mL of W5 dilution. The reactants were centrifuged at 300 rpm for 3 min to collect protoplasts and remove the supernatant. After washing twice, 1 mL of W5 solution was added again and cultured at 28 °C for 24 h in the dark. After transformation, GFP fluorescence was observed under a laser scanning confocal microscope (Nikon C2-ER, Japan). All transient expression assays were repeated at least four times.